protein recbyery



treatment.

Patented Au 10, 194

V a PRO-"ITE IN nno'ovnny- Lawrence W; Bass and Harold S. OlcottaPittsb u rgh,.- Pa., assignors to Cotton: Research Foundation, acorporation of Tennessee No Drawing. Application August 2, 1940, if i "SerialN0.350, 12?

This invention relates to therecovery of pro- 1 tein by precipitation from solution, andi'consists of a method that facilitates recovery, and 1 otherwise responsive to such reaches sources not .In Letters Patent" of the UnitedStatesNo.

to-nseed is dehulled andfragmented to the condition of'meal; from the meal the oil is first extracted, and with it the go-ssypo]; then, by leaching under specified conditions, the water-soluble protein is removed. The washed meal is dis persed in water (inthe presence of an antioxidant) and the dispersal is alkalinated to a pH value of 10. Under such conditions the desired protein goes into solution. The solution is separated from the solid residue; and from it by acidification the protein is precipitated. Nickerson says that, to effect this precipitation, he has employed hydrochloric, acetic, and sulfuric acids; and that he has found sulfuric to be best. He alludes to difiiculties attending acidification.

He says that the manner in which the, acid is added to the alkaline extract has considerable bearing upon the resulting precipitation; he

2,194,835; granted" on the application f Ralph g I". Nickerson, aprocedure is described of'the recovery of protein from cottonseed. The cotof carbon dioxide gas isjconveniently effected by bubbling the gas through in conventional ma nner. Withjth'eintroductiomof the gasthe excess alkali is neutralized, and'thezpH value of .the

solution: falls rapidly to 5.9-.-6.2. This is inthe "iso-electric region ofthe protein, and inthe region of' optimum precipitation; and precipitaa tionensues. E'Ihe" continuedfpassage of carbon dioxide gas into the solution I does; not reduce further the pI-Ivalue. Over-acidification does not occur; excessive local acid concentration does not occur; and the recoveryofthe protein'is notes the bad eifect of over-acidification; and

says that theaddition should proceed slowly, while the extract is stirred vigorously; otherwise, excessive local acid concentration may occur with accompanying denaturation of a portion of the protein. I

The present invention lies in the discovery that if, instead of acidification by the stirring in of a liquid acid such as sulfuric, the extract be acidified by bubbling with carbon dioxide gas into the solution not only will the desired precipitation be effected, but the procedure will.

be relieved of difiiculties of the nature indicated. The invention involves the further discovery that precipitation of protein from solution by saturation with carbon dioxide may, with proper control of circumstances, be extended to a wider range of utility.

In practising the invention uponcottonseed meal, the meal is first freed of its oil and gossypol contents, and its water-soluble protein content is leached away, as described in the Nickerson patent. It is then extracted with an alkali solution, and the alkaline extra-ct, carrying in solution the desired water-insolubl protein, is centrifuged free of the solids. Carbon dioxide gas is then introduced into and caused to satu-' rate the separated solution. This introduction A further important advantage of the rriethod resides in the production of proteins containing no adsorbed acid. When' proteins are precipitated from their solutions with strong acids such as sulphuric, the precipitate adsorbs large amounts of the acid. Such precipitates may be washed repeatedly with water without removing such acid. After drying, resuspension of the ground product in water yields a solution which may be even more acid (as measured by. pI-I) than the original precipitate. In the method of our invention, adsorption of acid may occur during the precipitation reactioni however, the acid (carbonic) is easily decomposed, yielding water andcarbon dioxide. Hence, when the protein solids are dried, the carbondioxide is dissipated in the air. Such proteins would in all likelihood be more stable on long standing than those prepared by inorganic acid precipitation, inasmuch as, in the latter case, the residual acid acts on standing in such manner as to denature the protein, render it less soluble in various reagents, and hence less useful in the arts.

Into an alkaline extract lbs.) of cottonseed meal, previously relieved of oil, gossypol, and water-soluble protein, carrying approximately three pounds of protein not soluble in water, and having a pH value of 10.4, carbon dioxide gas was introduced, using a bubbler of commercial type, at the rate of approximately 250 0. 0. per minute, and with the following effect upon pH value- Time in min. pH 7 It will be remarked on considering this tabulation that prolonged treatment with carbon dioxide did not have effect to reduce the pH value beyond 6.15, when once that 'value has been reached. And, as has been said, at that 'pH value precipitation occurs. 7

Similar resultsmay be reached with the meals of certain otherseeds of hempseed for example. The alkaline extract of hemps'eed meal may by "the introduction o'fcarbondioxide be brought to a pH value of substantially 6; and V at that pH value the globulin fraction of the 'hempseed protein '(edestin) will be precipitated I completely.v 7 But there remain other seeds whose alkaline extracts are not so immediately respon *sive to treatment with-carbon dioxide. Y

v the principal globulin of the soy-bean has an isoelectric region at pH 4.0 to 4.5.

--pH may be kept in the desired region. If the 1 pressure were released prior to the filtration operation, carbon dioxide would escape from the Our further invention lies in the discovery that} t by v'aryingcertain conditions under -which the L 7 carbon dioxide is introduced-the point toiwhich the pH value of a solution may be brought and at which it may'be'maintained can be varied;"

and .we-hav-e perceived that; by such variation,

the pH: values-thus attainable be} made sulge Q poincident withpthe iso-electrie point" io f the otherwise unresponsivesolution; whereupon precipitationjollowsf lve havediscovered 7 that suchvaria-tionmayinsomemeasure (though lightly, with n p missib e ran eibe'broueht about by change in temperature-and more efiec V tively by change of pressure. The .lowerthetemheraLturathe lower figure. to-Whichthe a value may-be reducedpthegreater thepressure,

.th i i e h w-t-r J a I J In the case of soy-bean protein, saturation with carbon dioxide at atmospheric-pressure does .not A result in precipitation. of -the protein from an alkaline extract of: the meal, Vinasm-uchas solution, the would rise to approximately 6.0,

=and re-solution would occur.

The method of the invention is applicable under atmospheric temperature and pressure to solutions whose protein precipitates at a pH value. of approximatelyfi; and is further applicable: under higher pressures to those whose pro- ..tein precipitates at a lower pH value than 6.

,1.Weclairn-asourinvention; p The vmethod herein described of recovering from oil-free cottonseed meal, after the .separ-ee tion from it of its water-soluble protein, its re- ,maining protein content, which .eonsists in extracting by means efan alkaline solvent such "remaining-protein content, and bubbling the solution through with carbon dioxide in gaseous statewhereby, oven-acidificatio being avoided, the pH value of -the1solution is brought within and maintained within the .iso-electrie region of the protein, ivith'rprecipita'tionof the protein.

AWRENCEW. Bass. rL-AR LI; s, onco'm". 

